This R21 proposal describes the development of three-dimensional (3-D) in vitro liver sinusoids comprised of primary hepatocytes and primary liver sinusoidal endothelial cells. These in vitro constructs will be used in detoxification studies. The specific goals of this proposal are 1. To design a novel polyelectrolyte-based scaffold for the sequential, 3-D layering of parenchymal (primary hepatocyte) and non-parenchymal (liver sinusoidal endothelial) cells and the optimization of hepatocyte-specific function. 2. To quantify cyochrome-P450 (CYP) enzyme efficiency, and, bile acid synthesis and composition in 3-D in vitro hepatic constructs. 3. To study detoxification and metabolism of the 3-D liver constructs under periportal and pericentral oxygen concentration. These cellular constructs will enable studies aimed at obtaining comprehensive information on the complex signaling pathways and cell-talk that occur in the liver during detoxification. Furthermore, such in vitro hepatic analogs will provide accurate models for immediate applications in bioreactor devices, disease pathogenesis, toxicity evaluations, and in the testing of pharmaceuticals and drugs. The methodology to layer cells outlined in this proposal is an emerging technology with enormous potential. The polyelectrolyte scaffold can be modified, both chemically, and physically to incorporate a diverse range of cell types. The proposed research, can in principle, be used to layer a wide range of disparate cell types, and therefore, will have a significant impact on tissue engineering in general, and hepatic tissue engineering in particular. The inherent versatility and simplicity of the methodology renders it suitable for translation into industrial tests and evaluations. PUBLIC HEALTH RELEVANCE: The proposed research will have a significant impact on tissue engineering in general, and hepatic tissue engineering in particular, by providing a versatile and robust technique to sequentially layer cells. The 3-D in vitro hepatocyte-liver sinusoidal endothelial cell constructs will provide physiologically-relevant information on liver detoxification and bile acid synthesis. These studies will form the basis for a comprehensive understanding of human-drug interactions.